Leishmaniasis, a multi-spectrum disease transmitted by sand fly vectors, has an important social-economic impact in many countries. A strong relationship exists between sand flies and Leishmania such that, in nature, only certain species of sand flies are able to transmit certain species of Leishmania. Such species-specificity is driven by several molecular factors that allow the parasite to infect, survive and multiply within the midgut of the sand fly and be transmitted to a suitable host during a blood meal. Some sand fly species are considered permissive in that they are able to harbor experimental infections of several Leishmania species (e.g. Lutzomyia. longipalpis);other sand fly species are considered restrictive as they can only be infected with the Leishmania species that they carry in nature (e.g. Phlebotomus. papatasi). The precise interactions that lead to this vector competence, whether for permissive or restrictive vectors, remain to be completely elucidated. We previously defined one specific interaction between L. major lipophosphoglycan and a P. papatasi galectin-like molecule that is essential for vector competence and demonstrated that antibodies targeted to this midgut molecule prevent establishment of Leishmania major within the sand fly midgut. Here we propose a set of studies using this restrictive vector/parasite system, to identify additional sand fly molecules that participate in the development of Leishmania within the sand fly midgut. Our studies will specifically focus on the use of RNA interference to assess the roles of proteins such as PpChym2, a blood-induced chymotrypsin that may be involved in early killing of Leishmania;and PpChit1, a midgut-specific chitinase that is thought to play a role in Leishmania escape from the peritrophic matrix. Additionally, recently identified midgut transcripts such as those coding for peritrophins and thrombin-specific inhibitors will also be evaluated. Once identified, P. papatasi molecules that effectively interfere with the development of L. major also will be tested for their broad spectrum activity in other sand fly vector combinations, including P. argentipes and L. donovani. This research will advance the knowledge regarding sand fly-Leishmania interaction and will provide insights for the selection of transmission blocking candidates as a strategy to eliminate and/or reduce leishmaniasis. Project Narrative: Phlebotomine sand flies are the main vectors of leishmaniasis, a multi-spectrum disease that causes substantial morbidity and mortality in the developing world. As effective therapies are un-affordable to most patients and no vaccines are available, vector-based strategies, specifically transmission-blocking vaccines are becoming a choice strategy to control many vector transmitted diseases. Our studies aim at assessing the effect of silencing a selected number of sand fly proteins thought to be involved in Leishmania development within the sand fly;elucidating how these proteins interact with Leishmania parasites undoubtedly will lead to the development of novel strategies to fight parasite transmission.